Help: PCR problem using pfu DNA pol

Stein Tore Solem steins at nfh.uit.no
Mon Oct 22 09:31:51 EST 2001


Hello

I am trying to amplify a specific cDNA from M-MLV RT reverse-transcribed
total RNA using pfu DNA polymerase. I have optimized the annealing temp.
using a regular Taq, and did not experience any problems while doing this.


However, when I switched to pfu DNA polymerase nothing was amplified at
all. pfu-amplification of the target cloned into a plasmid (100 pg plasmid
as template) gave PCR-products for some of my primer combinations,
suggesting that the activity of the enzyme is OK. The primers I am using
have 5'-extension that do not hybridize during the first cycle. The
reactions are performed as hot-start ie. I add the enzyme after the
template has denatured 1 min at 95°C. 

Why do I not get products using pfu DNA polymerase and cDNA as template?
Have I missed an important feature about pfu that could solve this
problem? I really appreciate any inputs on this one.

Stein Tore Solem
Norwegian College of Fishery Science
University of Tromsø
Norway




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